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Comprehensive profiling of transcriptional networks specific for lactogenic differentiation of HC11 mammary epithelial stem-like cells.

ABSTRACT: The development of mammary gland as a lactogenic tissue is a highly coordinated multistep process. The epithelial cells of lactiferous tubules undergo profound changes during the developmental window of puberty, pregnancy, and lactation. Several hormones including estrogen, progesterone, glucocorticoids and prolactin act in concert, and orchestrate the development of mammary gland. Understanding the gene regulatory networks that coordinate proliferation and differentiation of HC11 Mammary Epithelial stem-like Cells (MEC) under the influence of lactogenic hormones is critical for elucidating the mechanism of lactogenesis in detail. In this study, we analyzed transcriptome profiles of undifferentiated MEC (normal) and compared them with Murine Embryonic Stem Cells (ESC) using next-generation mRNA sequencing. Further, we analyzed the transcriptome output during lactogenic differentiation of MEC following treatment with glucocorticoids (primed state) and both glucocorticoids and prolactin together (prolactin state). We established stage-specific gene regulatory networks in ESC and MEC (normal, priming and prolactin states). We validated the top up-and downregulated genes in each stage of differentiation of MEC by RT-PCR and found that they are comparable with that of RNA-seq data. HC11 MEC display decreased expression of Pou5f1 and Sox2, which is crucial for the differentiation of MEC, which otherwise ensure pluripotency to ESC. Cited4 is induced during priming and is involved in milk secretion. MEC upon exposure to both glucocorticoids and prolactin undergo terminal differentiation, which is associated with the expression of several genes, including Xbp1 and Cbp that are required for cell growth and differentiation. Our study also identified differential expression of transcription factors and epigenetic regulators in each stage of lactogenic differentiation. We also analyzed the transcriptome data for the pathways that are selectively activated during lactogenic differentiation. Further, we found that selective expression of chromatin modulators (Dnmt3l, Chd9) in response to glucocorticoids suggests a highly coordinated stage-specific lactogenic differentiation of MEC.

SUBMITTER: Sornapudi TR

PROVIDER: S-EPMC6079013 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Comprehensive profiling of transcriptional networks specific for lactogenic differentiation of HC11 mammary epithelial stem-like cells.

Sornapudi Trinadha Rao TR Nayak Rakhee R Guthikonda Prashanth Kumar PK Pasupulati Anil Kumar AK Kethavath Srinivas S Uppada Vanita V Mondal Sukalpa S Yellaboina Sailu S Kurukuti Sreenivasulu S

Scientific reports 20180806 1

The development of mammary gland as a lactogenic tissue is a highly coordinated multistep process. The epithelial cells of lactiferous tubules undergo profound changes during the developmental window of puberty, pregnancy, and lactation. Several hormones including estrogen, progesterone, glucocorticoids and prolactin act in concert, and orchestrate the development of mammary gland. Understanding the gene regulatory networks that coordinate proliferation and differentiation of HC11 Mammary Epithe ...[more]

PMID: 30082875

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Project description:Purpose: Understanding of gene transcriptional networks specifying murine mammary epithelial cells (HC11) during lactogenic differentiation and its comparision with murine embryonic stem cells Methods: RNA-sequencing after ribosomal RNA depletion followed by cDNA synthesis, adaptor ligation and sequencing in duplicate of each sample using Illumina Hi-seq 2500 paired end sequencing. Sequence reads with adapters, over-represented and low-quality reads were discarded. High quality reads were mapped to reference mouse genome (mm10) using Bowtie followed derivation of FPKM values using cufflinks and DE-seq to observe differentially expressed genes. Results: Using standard analysis methods which were published recently we uniquely mapped more than 50 million sequence reads out of which total 11961 transcripts were from ESC, 11045 from Normal, 11243 from Primed and 10719 from prolactin stage. When we did DE-seq differential gene expression analysis we observed that for Normal to primed transition 1141 and 1026 transcripts were Up and Down regulated respectively where as upon transition from Primed to Prolactin stage 385 and 655 transcripts were Up and Down regulated respectively. We also did pathway analysis to interpret the transcript activation and its function during lactogeinc differentiation; we found that the pathways which are highly up regulated are related to carbohydrate, lipids, nucleic acid and protein metabolism. We also observed that most of the up regulated pathways were related to maintenance of mammary cells differentiated state and nutrition of the infants. Our study concludes that overall differentiation requires priming with glucocorticoids followed by prolactin stimulation of HC11 cells, which tunes the cells for the secretion of milk and its maintenance of the differentiated state till the lactation period. Conclusions: Transcriptome analysis by next generation sequencing allowed us to explore global gene expression during lactogenic differentiation of HC11 cells in presence of glucocortiocids in primed state. Glucocorticoids are activating genes, transcription factors, epigenetic modifiers and metabolic pathways which are required for later stages of lactation. On other hand gene expression analysis of prolactin stage revealed that there is a focused gene expression exclusively related to lactation process. We also observed that HC11 stem like marker are highly expressed in undifferentiated state whose expression was down regulated in primed and prolactin state indicating that HC11 are partially bipotent cells; when these cells receive differentiation signals they loose their stem cell like characteristics and are differentiated.

Project description:Retrotransposons copy their sequences via an RNA intermediate, followed by reverse transcription into cDNA and random insertion, into a new genomic locus. New retrotransposon copies may lead to cell transformation and/or tumorigenesis through insertional mutagenesis. Methylation is a major defense mechanism against retrotransposon RNA expression and retrotransposition in differentiated cells, whereas stem cells are relatively hypo?methylated. Epithelial?to?mesenchymal transition (EMT), which transforms normal epithelial cells into mesenchymal?like cells, also contributes to tumor progression and tumor metastasis. Cancer stem cells (CSCs), a fraction of undifferentiated tumor?initiating cancer cells, are reciprocally related to EMT. In the present study, the outcome of long terminal repeat (LTR)?Viral?Like 30 (VL30) retrotransposition was examined in mouse mammary stem?like/progenitor HC11 epithelial cells. The transfection of HC11 cells with a VL30 retrotransposon, engineered with an EGFP?based retrotransposition cassette, elicited a higher retrotransposition frequency in comparison to differentiated J3B1A and C127 mouse mammary cells. Fluorescence microscopy and PCR analysis confirmed the specificity of retrotransposition events. The differentiated retrotransposition?positive cells retained their epithelial morphology, while the respective HC11 cells acquired mesenchymal features associated with the loss of E?cadherin, the induction of N?cadherin, and fibronectin and vimentin protein expression, as well as an increased transforming growth factor (TGF)??1, Slug, Snail?1 and Twist mRNA expression. In addition, they were characterized by cell proliferation in low serum, and the acquisition of CSC?like properties indicated by mammosphere formation under anchorage?independent conditions. Mammospheres exhibited an increased Nanog and Oct4 mRNA expression and a CD44+/CD24?/low antigenic phenotype, as well as self?renewal and differentiation capacity, forming mammary acini?like structures. DNA sequencing analysis of retrotransposition?positive HC11 cells revealed retrotransposed VL30 copies integrated at the vicinity of EMT?, cancer type? and breast cancer?related genes. The inoculation of these cells into Balb/c mice produced cytokeratin?positive tumors containing pancytokeratin?positive cells, indicative of cell invasion features. On the whole, the findings of the present study demonstrate, for the first time, to the best of our knowledge, that stem?like epithelial HC11 cells are amenable to VL30 retrotransposition associated with the induction of EMT and CSC generation, leading to tumorigenesis.

Dataset Information

Comprehensive profiling of transcriptional networks specific for lactogenic differentiation of HC11 mammary epithelial stem-like cells.

Publications

Comprehensive profiling of transcriptional networks specific for lactogenic differentiation of HC11 mammary epithelial stem-like cells.

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